Colony stimulating factors (CSFs) which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. CSFs in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G-CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and macrophage colonies, respectively, while GM-CSF and interleukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies. IL-3 also stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies.
U.S. Pat. No. 4,877,729 and U.S. Pat. No. 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.
International Patent Application (PCT) WO 88/00598 discloses gibbon- and human-like IL-3. The hIL-3 contains a Ser.sup.8 -&gt;Pro.sup.8 replacement. Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.
U.S. Pat. No. 4,810,643 discloses the DNA sequence encoding human G-CSF.
WO 91/02754 discloses a fusion protein comprised of GM-CSF and IL-3 which has increased biological activity compared to GM-CSF or IL-3 alone. Also disclosed are nonglycosylated IL-3 and GM-CSF analog proteins as components of the multi-functional hematopoietic receptor agonist.
WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and G-CSF.
WO 95/21197 and WO 95/21254 disclose fusion proteins capable of broad multi-functional hematopoietic properties.
GB 2,285,446 relates to the c-mpl ligand (thrombopoietin) and various forms of thrombopoietin which are shown to influence the replication, differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.
EP 675,201 A1 relates to the c-mpl ligand (Megakaryocyte growth and development factor (MGDF), allelic variations of c-mpl ligand and c-mpl ligand attached to water soluble polymers such as polyethylene glycol.
WO 95/21920 provides the murine and human c-mpl ligand and polypeptide fragments thereof. The proteins are useful for in vivo and ex vivo therapy for stimulating platelet production.
Rearrangement of Protein Sequences
In evolution, rearrangements of DNA sequences serve an important role in generating a diversity of protein structure and function. Gene duplication and exon shuffling provide an important mechanism to rapidly generate diversity and thereby provide organisms with a competitive advantage, especially since the basal mutation rate is low (Doolittle, Protein Science 1: 191-200, 1992).
The development of recombinant DNA methods has made it possible to study the effects of sequence transposition on protein folding, structure and function. The approach used in creating new sequences resembles that of naturally occurring pairs of proteins that are related by linear reorganization of their amino acid sequences (Cunningham, et al., Proc. Natl. Acad. Sci. U.S.A. 76: 3218-3222, 1979; Teather & Erfle, J. Bacteriol. 172: 3837-3841, 1990; Schimming et al., Eur. J. Biochem. 204: 13-19, 1992; Yamiuchi and Minamikawa, FEBS Lett. 260: 127-130, 1991; MacGregor et al., FEBS Lett. 378: 263-266). The first in vitro application of this type of rearrangement to proteins was described by Goldenberg and Creighton (J. Mol. Biol. 165: 407-413, 1983). A new N-terminus is selected at an internal site (breakpoint) of the original sequence, the new sequence having the same order of amino acids as the original from the breakpoint until it reaches an amino acid that is at or near the original C-terminus. At this point the new sequence is joined, either directly or through an additional portion of sequence (linker), to an amino acid that is at or near the original N-terminus, and the new sequence continues with the same sequence as the original until it reaches a point that is at or near the amino acid that was N-terminal to the breakpoint site of the original sequence, this residue forming the new C-terminus of the chain.
This approach has been applied to proteins which range in size from 58 to 462 amino acids (Goldenberg & Creighton, J. Mol. Biol. 165: 407-413, 1983; Li & Coffino, Mol. Cell. Biol. 13: 2377-2383, 1993). The proteins examined have represented a broad range of structural classes, including proteins that contain predominantly .alpha.-helix (interleukin-4; Kreitman et al., Cytokine 7: 311-318, 1995), .beta.-sheet (interleukin-1; Horlick et al., Protein Eng. 5: 427-431, 1992), or mixtures of the two (yeast phosphoribosyl anthranilate isomerase; Luger et al., Science 243: 206-210, 1989). Broad categories of protein function are represented in these sequence reorganization studies:
Enzymes
T4 lysozyme Zhang et al., Biochemistry 32: 12311-12318, 1993; Zhang et al., Nature Struct. Biol. 1: 434-438 (1995) PA0 dihydrofolate reductase Buchwalder et al., Biochemistry 31: 1621-1630, 1994; Protasova et al., Prot. Eng. 7: 1373-1377, 1995) PA0 ribonuclease T1 Mullins et al., J. Am. Chem. Soc. 116: 5529-5533, 1994; Garrett et al., Protein Science 5: 204-211, 1996) PA0 Bacillus .beta.-glucanse Hahn et al., Proc. Natl. Acad. Sci. U.S.A. 91: 10417-10421, 1994) PA0 aspartate transcarbamoylase Yang & Schachman, Proc. Natl. Acad. Sci. U.S.A. 90: 11980-11984, 1993) PA0 phosphoribosyl anthranilate isomerase Luger et al., Science 243: 206-210 (1989; Luger et al., Prot. Eng. 3: 249-258, 1990) PA0 pepsin/pepsinogen Lin et al., Protein Science 4: 159-166, 1995) PA0 glyceraldehyde-3-phosphate dehydrogenase Vignais et al., Protein Science 4: 994-1000, 1995) PA0 ornithine decarboxylase Li & Coffino, Mol. Cell. Biol. 13: 2377-2383, 1993) PA0 yeast phosphoglycerate dehydrogenase Ritco-Vonsovici et al., Biochemistry 34: 16543-16551, 1995) PA0 basic pancreatic trypsin inhibitor Goldenberg & Creighton, J. Mol. Biol. 165: 407-413, 1983) PA0 interleukin-1.beta. Horlick et al., Protein Eng. 5: 427-431, 1992) PA0 interleukin-4 Kreitman et al., Cytokine 7: 311-318, 1995) PA0 .alpha.-spectrin SH3 domain Viguera, et al., J. Mol. Biol. 247: 670-681, 1995) PA0 omp A Koebnik & Kramer, J. Mol. Biol. 250: 617-626, 1995) PA0 interleukin-4-Pseudomonas exotoxin Kreitman et al., Proc. Natl. Acad. Sci. U.S.A. 91: 6889-6893, 1994). PA0 Xaa at position 1 is Thr, Ser, Arg, Tyr or Gly; PA0 Xaa at position 2 is Pro or Leu; PA0 Xaa at position 3 is Leu, Arg, Tyr or Ser; PA0 Xaa at position 13 is Phe, Ser, His, Thr or Pro; PA0 Xaa at position 16 is Lys, Pro, Ser, Thr or His; PA0 Xaa at position 17 is Cys, Ser, Gly, Ala, Ile, Tyr or Arg; PA0 Xaa at position 18 is Leu, Thr, Pro, His, Ile or Cys; PA0 Xaa at position 22 is Arg, Tyr, Ser, Thr or Ala; PA0 Xaa at position 24 is Ile, Pro, Tyr or Leu; PA0 Xaa at position 27 is Asp, or Gly; PA0 Xaa at position 30 is Ala, Ile, Leu or Gly; PA0 Xaa at position 34 is Lys or Ser; PA0 Xaa at position 36 is Cys or Ser; PA0 Xaa at position 42 is Cys or Ser; PA0 Xaa at position 43 is His, Thr, Gly, Val, Lys, Trp, Ala, Arg, Cys, or Leu; PA0 Xaa at position 44 is Pro, Gly, Arg, Asp, Val, Ala, His, Trp, Gln, or Thr; PA0 Xaa at position 46 is Glu, Arg, Phe, Arg, Ile or Ala; PA0 Xaa at position 47 is Leu or Thr; PA0 Xaa at position 49 is Leu, Phe, Arg or Ser; PA0 Xaa at position 50 is Leu, Ile, His, Pro or Tyr; PA0 Xaa at position 54 is Leu or His; PA0 Xaa at position 64 is Cys or Ser; PA0 Xaa at position 67 is Gln, Lys, Leu or Cys; PA0 Xaa at position 70 is Gln, Pro, Leu, Arg or Ser; PA0 Xaa at position 74 is Cys or Ser; PA0 Xaa at position 104 is Asp, Gly or Val; PA0 Xaa at position 108 is Leu, Ala, Val, Arg, Trp, Gln or Gly; PA0 Xaa at position 115 is Thr, His, Leu or Ala; PA0 Xaa at position 120 is Gln, Gly, Arg, Lys or His PA0 Xaa at position 123 is Glu, Arg, Phe or Thr PA0 Xaa at position 144 is Phe, His, Arg, Pro, Leu, Gln or Glu; PA0 Xaa at position 146 is Arg or Gln; PA0 Xaa at position 147 is Arg or Gln; PA0 Xaa at position 156 is His, Gly or Ser; PA0 Xaa at position 159 is Ser, Arg, Thr, Tyr, Val or Gly; PA0 Xaa at position 162 is Glu, Leu, Gly or Trp; PA0 Xaa at position 163 is Val, Gly, Arg or Ala; PA0 Xaa at position 169 is Arg, Ser, Leu, Arg or Cys; PA0 Xaa at position 170 is His, Arg or Ser; PA0 wherein optionally 1-11 amino acids from the N-terminus and 1-5 from the C-terminus can be deleted; and PA0 wherein the N-terminus is joined to the C-terminus directly or through a linker capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids; PA0 Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; PA0 Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; PA0 Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; PA0 Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; PA0 Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; PA0 Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; PA0 Xaa at position 23 is Ile, Val, Ala, Gly, Trp, Lys, Phe, Leu, Ser, or Arg; PA0 Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; PA0 Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; PA0 Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; PA0 Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; PA0 Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; PA0 Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; PA0 Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; PA0 Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; PA0 Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; PA0 Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; PA0 Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; PA0 Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; PA0 Xaa at position 36 is Asp, Leu, or Val; PA0 Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; PA0 Xaa at position 38 is Asn, or Ala; PA0 Xaa at position 40 is Leu, Trp, or Arg; PA0 Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; PA0 Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; PA0 Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; PA0 Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; PA0 Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; PA0 Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; PA0 Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; PA0 Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; PA0 Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; PA0 Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; PA0 Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; PA0 Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; PA0 Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or Met; PA0 Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; PA0 Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; PA0 Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; PA0 Xaa at position 57 is Asn or Gly; PA0 Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; PA0 Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; PA0 Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; PA0 Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; PA0 Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; PA0 Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; PA0 Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; PA0 Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; PA0 Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; PA0 Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; PA0 Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; PA0 Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; PA0 Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; PA0 Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; PA0 Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; PA0 Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; PA0 Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; PA0 Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; PA0 Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; PA0 Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; PA0 Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; PA0 Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; PA0 Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; PA0 Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; PA0 Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; PA0 Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; PA0 Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; PA0 Xaa at position 85 is Leu, Asn, Val, or Gln; PA0 Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; PA0 Xaa at position 87 is Leu, Ser, Trp, or Gly; PA0 Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; PA0 Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; PA0 Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; PA0 Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; PA0 Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; PA0 Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; PA0 Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; PA0 Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; PA0 Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; PA0 Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; PA0 Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; PA0 Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; PA0 Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; PA0 Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln; PA0 Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; PA0 Xaa at position 103 is Asp, or Ser; PA0 Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; PA0 Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; PA0 Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; PA0 Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; PA0 Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; PA0 Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; PA0 Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; PA0 Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; PA0 Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; PA0 Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; PA0 Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; PA0 Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; PA0 Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; PA0 Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; PA0 Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; PA0 Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; PA0 Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; PA0 Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; PA0 Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; PA0 wherein optionally from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from 0 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; and PA0 wherein the N-terminus is joined to the C-terminus directly or through a linker (L.sub.2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids; PA0 Xaa at position 112 is deleted or Leu, Ala, Val, Ile, Pro, Phe, Trp, or Met; PA0 Xaa at position 113 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; PA0 Xaa at position 114 is deleted or Pro, Phe, Ala, Val, Leu, Ile, Trp, or Met; PA0 Xaa at position 115 is deleted or Gln, Gly, Ser, Thr, Tyr, or Asn; and PA0 wherein the N-terminus is joined to the C-terminus directly or through a linker (L.sub.2) capable of joining the N-terminus to the C-terminus and having new C- and N-termini at amino acids; PA0 Xaa at position 17 is Ser, Lys, Gly, Asp, Met, Gln, or Arg; PA0 Xaa at position 18 is Asn, His, Leu, Ile, Phe, Arg, or Gln; PA0 Xaa at position 19 is Met, Phe, Ile, Arg, Gly, Ala, or Cys; PA0 Xaa at position 20 is Ile, Cys, Gln, Glu, Arg, Pro, or Ala; PA0 Xaa at position 21 is Asp, Phe, Lys, Arg, Ala, Gly, Glu, Gln, Asn, Thr, Ser or Val; PA0 Xaa at position 22 is Glu, Trp, Pro, Ser, Ala, His, Asp, Asn, Gln, Leu, Val or Gly; PA0 Xaa at position 23 is Ile, Val, Ala, Gly, Trp, Lys, Phe, Leu, Ser, or Arg; PA0 Xaa at position 24 is Ile, Gly, Val, Arg, Ser, Phe, or Leu; PA0 Xaa at position 25 is Thr, His, Gly, Gln, Arg, Pro, or Ala; PA0 Xaa at position 26 is His, Thr, Phe, Gly, Arg, Ala, or Trp; PA0 Xaa at position 27 is Leu, Gly, Arg, Thr, Ser, or Ala; PA0 Xaa at position 28 is Lys, Arg, Leu, Gln, Gly, Pro, Val or Trp; PA0 Xaa at position 29 is Gln, Asn, Leu, Pro, Arg, or Val; PA0 Xaa at position 30 is Pro, His, Thr, Gly, Asp, Gln, Ser, Leu, or Lys; PA0 Xaa at position 31 is Pro, Asp, Gly, Ala, Arg, Leu, or Gln; PA0 Xaa at position 32 is Leu, Val, Arg, Gln, Asn, Gly, Ala, or Glu; PA0 Xaa at position 33 is Pro, Leu, Gln, Ala, Thr, or Glu; PA0 Xaa at position 34 is Leu, Val, Gly, Ser, Lys, Glu, Gln, Thr, Arg, Ala, Phe, Ile or Met; PA0 Xaa at position 35 is Leu, Ala, Gly, Asn, Pro, Gln, or Val; PA0 Xaa at position 36 is Asp, Leu, or Val; PA0 Xaa at position 37 is Phe, Ser, Pro, Trp, or Ile; PA0 Xaa at position 38 is Asn, or Ala; PA0 Xaa at position 40 is Leu, Trp, or Arg; PA0 Xaa at position 41 is Asn, Cys, Arg, Leu, His, Met, or Pro; PA0 Xaa at position 42 is Gly, Asp, Ser, Cys, Asn, Lys, Thr, Leu, Val, Glu, Phe, Tyr, Ile, Met or Ala; PA0 Xaa at position 43 is Glu, Asn, Tyr, Leu, Phe, Asp, Ala, Cys, Gln, Arg, Thr, Gly or Ser; PA0 Xaa at position 44 is Asp, Ser, Leu, Arg, Lys, Thr, Met, Trp, Glu, Asn, Gln, Ala or Pro; PA0 Xaa at position 45 is Gln, Pro, Phe, Val, Met, Leu, Thr, Lys, Trp, Asp, Asn, Arg, Ser, Ala, Ile, Glu or His; PA0 Xaa at position 46 is Asp, Phe, Ser, Thr, Cys, Glu, Asn, Gln, Lys, His, Ala, Tyr, Ile, Val or Gly; PA0 Xaa at position 47 is Ile, Gly, Val, Ser, Arg, Pro, or His; PA0 Xaa at position 48 is Leu, Ser, Cys, Arg, Ile, His, Phe, Glu, Lys, Thr, Ala, Met, Val or Asn; PA0 Xaa at position 49 is Met, Arg, Ala, Gly, Pro, Asn, His, or Asp; PA0 Xaa at position 50 is Glu, Leu, Thr, Asp, Tyr, Lys, Asn, Ser, Ala, Ile, Val, His, Phe, Met or Gln; PA0 Xaa at position 51 is Asn, Arg, Met, Pro, Ser, Thr, or His; PA0 Xaa at position 52 is Asn, His, Arg, Leu, Gly, Ser, or Thr; PA0 Xaa at position 53 is Leu, Thr, Ala, Gly, Glu, Pro, Lys, Ser, or PA0 Xaa at position 54 is Arg, Asp, Ile, Ser, Val, Thr, Gln, Asn, Lys, His, Ala or Leu; PA0 Xaa at position 55 is Arg, Thr, Val, Ser, Leu, or Gly; PA0 Xaa at position 56 is Pro, Gly, Cys, Ser, Gln, Glu, Arg, His, Thr, Ala, Tyr, Phe, Leu, Val or Lys; PA0 Xaa at position 57 is Asn or Gly; PA0 Xaa at position 58 is Leu, Ser, Asp, Arg, Gln, Val, or Cys; PA0 Xaa at position 59 is Glu Tyr, His, Leu, Pro, or Arg; PA0 Xaa at position 60 is Ala, Ser, Pro, Tyr, Asn, or Thr; PA0 Xaa at position 61 is Phe, Asn, Glu, Pro, Lys, Arg, or Ser; PA0 Xaa at position 62 is Asn, His, Val, Arg, Pro, Thr, Asp, or Ile; PA0 Xaa at position 63 is Arg, Tyr, Trp, Lys, Ser, His, Pro, or Val; PA0 Xaa at position 64 is Ala, Asn, Pro, Ser, or Lys; PA0 Xaa at position 65 is Val, Thr, Pro, His, Leu, Phe, or Ser; PA0 Xaa at position 66 is Lys, Ile, Arg, Val, Asn, Glu, or Ser; PA0 Xaa at position 67 is Ser, Ala, Phe, Val, Gly, Asn, Ile, Pro, or His; PA0 Xaa at position 68 is Leu, Val, Trp, Ser, Ile, Phe, Thr, or His; PA0 Xaa at position 69 is Gln, Ala, Pro, Thr, Glu, Arg, Trp, Gly, or Leu; PA0 Xaa at position 70 is Asn, Leu, Val, Trp, Pro, or Ala; PA0 Xaa at position 71 is Ala, Met, Leu, Pro, Arg, Glu, Thr, Gln, Trp, or Asn; PA0 Xaa at position 72 is Ser, Glu, Met, Ala, His, Asn, Arg, or Asp; PA0 Xaa at position 73 is Ala, Glu, Asp, Leu, Ser, Gly, Thr, or Arg; PA0 Xaa at position 74 is Ile, Met, Thr, Pro, Arg, Gly, Ala; PA0 Xaa at position 75 is Glu, Lys, Gly, Asp, Pro, Trp, Arg, Ser, Gln, or Leu; PA0 Xaa at position 76 is Ser, Val, Ala, Asn, Trp, Glu, Pro, Gly, or Asp; PA0 Xaa at position 77 is Ile, Ser, Arg, Thr, or Leu; PA0 Xaa at position 78 is Leu, Ala, Ser, Glu, Phe, Gly, or Arg; PA0 Xaa at position 79 is Lys, Thr, Asn, Met, Arg, Ile, Gly, or Asp; PA0 Xaa at position 80 is Asn, Trp, Val, Gly, Thr, Leu, Glu, or Arg; PA0 Xaa at position 81 is Leu, Gln, Gly, Ala, Trp, Arg, Val, or Lys; PA0 Xaa at position 82 is Leu, Gln, Lys, Trp, Arg, Asp, Glu, Asn, His, Thr, Ser, Ala, Tyr, Phe, Ile, Met or Val; PA0 Xaa at position 83 is Pro, Ala, Thr, Trp, Arg, or Met; PA0 Xaa at position 84 is Cys, Glu, Gly, Arg, Met, or Val; PA0 Xaa at position 85 is Leu, Asn, Val, or Gln; PA0 Xaa at position 86 is Pro, Cys, Arg, Ala, or Lys; PA0 Xaa at position 87 is Leu, Ser, Trp, or Gly; PA0 Xaa at position 88 is Ala, Lys, Arg, Val, or Trp; PA0 Xaa at position 89 is Thr, Asp, Cys, Leu, Val, Glu, His, Asn, or Ser; PA0 Xaa at position 90 is Ala, Pro, Ser, Thr, Gly, Asp, Ile, or Met; PA0 Xaa at position 91 is Ala, Pro, Ser, Thr, Phe, Leu, Asp, or His; PA0 Xaa at position 92 is Pro, Phe, Arg, Ser, Lys, His, Ala, Gly, Ile or Leu; PA0 Xaa at position 93 is Thr, Asp, Ser, Asn, Pro, Ala, Leu, or Arg; PA0 Xaa at position 94 is Arg, Ile, Ser, Glu, Leu, Val, Gln, Lys, His, Ala, or Pro; PA0 Xaa at position 95 is His, Gln, Pro, Arg, Val, Leu, Gly, Thr, Asn, Lys, Ser, Ala, Trp, Phe, Ile, or Tyr; PA0 Xaa at position 96 is Pro, Lys, Tyr, Gly, Ile, or Thr; PA0 Xaa at position 97 is Ile, Val, Lys, Ala, or Asn; PA0 Xaa at position 98 is His, Ile, Asn, Leu, Asp, Ala, Thr, Glu, Gln, Ser, Phe, Met, Val, Lys, Arg, Tyr or Pro; PA0 Xaa at position 99 is Ile, Leu, Arg, Asp, Val, Pro, Gln, Gly, Ser, Phe, or His; PA0 Xaa at position 100 is Lys, Tyr, Leu, His, Arg, Ile, Ser, Gln, or Pro; PA0 Xaa at position 101 is Asp, Pro, Met, Lys, His, Thr, Val, Tyr, Glu, Asn, Ser, Ala, Gly, Ile, Leu, or Gln; PA0 Xaa at position 102 is Gly, Leu, Glu, Lys, Ser, Tyr, or Pro; PA0 Xaa at position 103 is Asp, or Ser; PA0 Xaa at position 104 is Trp, Val, Cys, Tyr, Thr, Met, Pro, Leu, Gln, Lys, Ala, Phe, or Gly; PA0 Xaa at position 105 is Asn, Pro, Ala, Phe, Ser, Trp, Gln, Tyr, Leu, Lys, Ile, Asp, or His; PA0 Xaa at position 106 is Glu, Ser, Ala, Lys, Thr, Ile, Gly, or Pro; PA0 Xaa at position 108 is Arg, Lys, Asp, Leu, Thr, Ile, Gln, His, Ser, Ala or Pro; PA0 Xaa at position 109 is Arg, Thr, Pro, Glu, Tyr, Leu, Ser, or Gly; PA0 Xaa at position 110 is Lys, Ala, Asn, Thr, Leu, Arg, Gln, His, Glu, Ser, or Trp; PA0 Xaa at position 111 is Leu, Ile, Arg, Asp, or Met; PA0 Xaa at position 112 is Thr, Val, Gln, Tyr, Glu, His, Ser, or Phe; PA0 Xaa at position 113 is Phe, Ser, Cys, His, Gly, Trp, Tyr, Asp, Lys, Leu, Ile, Val or Asn; PA0 Xaa at position 114 is Tyr, Cys, His, Ser, Trp, Arg, or Leu; PA0 Xaa at position 115 is Leu, Asn, Val, Pro, Arg, Ala, His, Thr, Trp, or Met; PA0 Xaa at position 116 is Lys, Leu, Pro, Thr, Met, Asp, Val, Glu, Arg, Trp, Ser, Asn, His, Ala, Tyr, Phe, Gln, or Ile; PA0 Xaa at position 117 is Thr, Ser, Asn, Ile, Trp, Lys, or Pro; PA0 Xaa at position 118 is Leu, Ser, Pro, Ala, Glu, Cys, Asp, or Tyr; PA0 Xaa at position 119 is Glu, Ser, Lys, Pro, Leu, Thr, Tyr, or Arg; PA0 Xaa at position 120 is Asn, Ala, Pro, Leu, His, Val, or Gln; PA0 Xaa at position 121 is Ala, Ser, Ile, Asn, Pro, Lys, Asp, or Gly; PA0 Xaa at position 122 is Gln, Ser, Met, Trp, Arg, Phe, Pro, His, Ile, Tyr, or Cys; PA0 Xaa at position 123 is Ala, Met, Glu, His, Ser, Pro, Tyr, or Leu; PA0 wherein optionally from 1 to 14 amino acids can be deleted from the N-terminus and/or from 1 to 15 amino acids can be deleted from the C-terminus; and wherein from 1 to 44 of the amino acids designated by Xaa are different from the corresponding amino acids of native (1-133) human interleukin-3; or PA0 and wherein L.sub.1 is a linker capable of linking R.sub.1 to R.sub.2 ;
Enzyme Inhibitor
Cytokines
Tyrosine Kinase Recognition Domain
Transmembrane Protein
Chimeric Protein
The results of these studies have been highly variable. In many cases substantially lower activity, solubility or thermodynamic stability were observed (E. coli dihydrofolate reductase, aspartate transcarbamoylase, phosphoribosyl anthranilate isomerase, glyceraldehyde-3-phosphate dehydrogenase, ornithine decarboxylase, omp A, yeast phosphoglycerate dehydrogenase). In other cases, the sequence rearranged protein appeared to have many nearly identical properties as its natural counterpart (basic pancreatic trypsin inhibitor, T4 lysozyme, ribonuclease T1, Bacillus .beta.-glucanase, interleukin-1.beta., .alpha.-spectrin SH3 domain, pepsinogen, interleukin-4). In exceptional cases, an unexpected improvement over some properties of the natural sequence was observed, e.g., the solubility and refolding rate for rearranged .alpha.-spectrin SH3 domain sequences, and the receptor affinity and anti-tumor activity of transposed interleukin-4-Pseudomonas exotoxin fusion molecule (Kreitman et al., Proc. Natl. Acad. Sci. U.S.A. 91: 6889-6893, 1994; Kreitman et al., Cancer Res. 55: 3357-3363, 1995).
The primary motivation for these types of studies has been to study the role of short-range and long-range interactions in protein folding and stability. Sequence rearrangements of this type convert a subset of interactions that are long-range in the original sequence into short-range interactions in the new sequence, and vice versa. The fact that many of these sequence rearrangements are able to attain a conformation with at least some activity is persuasive evidence that protein folding occurs by multiple folding pathways (Viguera, et al., J. Mol. Biol. 247: 670-681, 1995). In the case of the SH3 domain of a-spectrin, choosing new termini at locations that corresponded to .beta.-hairpin turns resulted in proteins with slightly less stability, but which were nevertheless able to fold.
The positions of the internal breakpoints used in the studies cited here are found exclusively on the surface of proteins, and are distributed throughout the linear sequence without any obvious bias towards the ends or the middle (the variation in the relative distance from the original N-terminus to the breakpoint is ca. 10 to 80% of the total sequence length). The linkers connecting the original N- and C-termini in these studies have ranged from 0 to 9 residues. In one case (Yang & Schachman, Proc. Natl. Acad. Sci. U.S.A. 90: 11980-11984, 1993), a portion of sequence has been deleted from the original C-terminal segment, and the connection made from the truncated C-terminus to the original N-terminus. Flexible hydrophilic residues such as Gly and Ser are frequently used in the linkers. Viguera, et al. (J. Mol. Biol. 247: 670-681, 1995) compared joining the original N- and C-termini with 3- or 4-residue linkers; the 3-residue linker was less thermodynamically stable. Protasova et al. (Protein Eng. 7: 1373-1377, 1994) used 3- or 5-residue linkers in connecting the original N-termini of E. coli dihydrofolate reductase; only the 3-residue linker produced protein in good yield.